Methods and compositions for the treatment of eye disorders with increased intraocular pressure

ABSTRACT

The present invention relates to methods and compositions that decrease intraocular pressure (IOP) of the eye. The compositions of the invention comprise short interfering nucleic acid molecules (siNA) including, but not limited to, siRNA that decrease expression of genes associated with production or drainage of intraocular fluid. The compositions of the invention can be used in the preparation of a medicament for the treatment of an eye conditions displaying increased IOP such as glaucoma, infection, inflammation, uveitis, and diabetic retinopathy. The methods of the invention comprise the administration to a patient in need thereof an effective amount of one or more siNAs of the invention.

This application is a divisional of application Ser. No. 11/360,305filed Feb. 22, 2006, issued Sep. 22, 2009 as U.S. Pat. No. 7,592,325which is continuation-in-part of International Patent Application No.PCT/GB2005/050134, filed Aug. 23, 2005, which claims priority to BritishApplication No. GB0503412.9, filed Feb. 18, 2005, and to BritishApplication No. GB0418762.1, filed Aug. 23, 2004, the contents of eachof which are incorporated in their entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions that decreaseintraocular pressure (IOP) of the eye. The compositions of the inventioncomprise short interfering nucleic acid molecules (siNA) including, butnot limited to, siRNA that decrease expression of genes associated withproduction or drainage of intraocular fluid. The compositions of theinvention can be used in the preparation of a medicament for thetreatment of an eye conditions displaying increased IOP such asglaucoma, infection, inflammation, uveitis, and diabetic retinopathy.The methods of the invention comprise the administration to a patient inneed thereof an effective amount of one or more siNAs of the invention.

2. BACKGROUND OF THE INVENTION

Glaucoma is one of the leading causes of blindness. Approximately 15% ofcases of blindness world-wide result from glaucoma. The most commontype, primary open-angle glaucoma, has a prevalence of 1/200 in thegeneral population over 40 years of age. Glaucoma has been simplydefined as the process of ocular tissue destruction caused by asustained elevation of the Intra Ocular Pressure (IOP) above its normalphysiological limits. Although several etiologies may be involved in theglaucoma complex, an absolute determinant in therapy selection is theamount of primary and/or induced change in pressure within theiridocorneal angle.

Current therapies include medications or surgeries aimed at loweringthis pressure, although the pathophysiological mechanisms by whichelevated IOP leads to neuronal damage in glaucoma are unknown. Medicalsuppression of an elevated IOP can be attempted using four types ofdrugs: (1) the aqueous humor formation suppressors (such as carbonicanhydrase inhibitors, beta-adrenergic blocking agents, andalpha2-adrenoreceptor agonists); (2) miotics (such asparasympathomimetics, including cholinergics and anticholinesteraseinhibitors); (3) uveoscleral outflow enhancers; and (4) hyperosmoticagents (that produce an osmotic pressure gradient across theblood/aqueous barrier within the cilliary epithelium). A fifth categoryof drugs, neuroprotection agents, is emerging as an important additionto medical therapy, including, for example, NOS inhibitors, excitatoryamino acid antagonists, glutamate receptor antagonists, apoptosisinhibitors, and calcium channel blockers.

Reviews of various eye disorders and their treatments can be found inthe following references: Bunce et al., 2005, Graefes Arch Clin ExpOphthalmol.; 243(4):294; Costagliola et al., 2000, Exp Eye Res.71(2):167; Costagliola et al., 1995, Eur J Ophthalmol., 5(1):19;Cullinane et al., 2002, Br J Ophthalmol., 86(6):676; Sakaguchi et al.,2002, Curr Eye Res. 24(5):325; Shah et al., 2000, J CardiovascPharmacol., 36(2):169, and Wang et al., 2005, Exp Eye Res., 80(5):629.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing mediated by short interfering RNAs(siRNA). After the discovery of the phenomenon in plants in the early1990s, Andy Fire and Craig Mello demonstrated that double-stranded RNA(dsRNA) specifically and selectively inhibited gene expression in anextremely efficient manner in Caenorhabditis elegans (Fire et al., 1998,Nature, 391:806). The sequence of the first strand (sense RNA) coincidedwith that of the corresponding region of the target messenger RNA(mRNA). The second strand (antisense RNA) was complementary to the mRNA.The resulting dsRNA turned out to be several orders of magnitude moreefficient than the corresponding single-stranded RNA molecules (inparticular, antisense RNA).

The process of RNAi begins when the enzyme, DICER, encounters dsRNA andchops it into pieces called small-interfering RNAs (siRNA). This proteinbelongs to the RNase III nuclease family. A complex of proteins gathersup these RNA remains and uses their code as a guide to search out anddestroy any RNAs in the cell with a matching sequence, such as targetMRNA (see Bosher & Labouesse, 2000, Nat Cell Biol, 2000, 2(2):E31, andAkashi et al., 2001, Antisense Nucleic Acid Drug Dev, 11(6):359).

In attempting to utilize RNAi for gene knockdown, it was recognized thatmammalian cells have developed various protective mechanisms againstviral infections that could impede the use of this approach. Indeed, thepresence of extremely low levels of viral dsRNA triggers an interferonresponse, resulting in a global non-specific suppression of translation,which in turn triggers apoptosis (Williams, 1997, Biochem Soc Trans,25(2):509; Gil & Esteban, 2000, Apoptosis, 5(2):107-14).

In 2000 dsRNA was reported to specifically inhibit 3 genes in the mouseoocyte and early embryo. Translational arrest, and thus a PKR response,was not observed as the embryos continued to develop (Wianny &Zernicka-Goetz, 2000, Nat Cell Biol, 2(2):70). Research at Ribopharma AG(Kulmbach, Germany) demonstrated the functionality of RNAi in mammaliancells, using short (20-24 base pairs) dsRNA to switch off genes in humancells without initiating the acute-phase response. Similar experimentscarried out by other research groups confirmed these results. (Elbashiret al., 2001, Genes Dev, 15(2):188; Caplen et al., 2001, Proc. Natl.Acad. Sci. USA, 98:9742) Tested in a variety of normal and cancer humanand mouse cell lines, it was determined that short hairpin RNAs (shRNA)can silence genes as efficiently as their siRNA counterparts (Paddisonet al, 2002, Genes Dev, 16(8):948). Recently, another group of smallRNAs (21-25 base pairs) was shown to mediate downregulation of geneexpression. These RNAs, small temporally regulated RNAs (stRNA),regulate timing of gene expression during development in Caenorhabditiselegans. (for review see Banerjee & Slack, 2002 and Grosshans & Slack,2002, J Cell Biol, 156(1):17).

Scientists have used RNAi in several systems, including Caenorhabditiselegans, Drosophila, trypanosomes, and other invertebrates. Severalgroups have recently presented the specific suppression of proteinbiosynthesis in different mammalian cell lines (specifically in HeLacells) demonstrating that RNAi is a broadly applicable method for genesilencing in vitro. Based on these results, RNAi has rapidly become awell recognized tool for validating (identifying and assigning) genefunction. RNAi employing short dsRNA oligonucleotides will yield anunderstanding of the function of genes that are only partiallysequenced.

The preceding is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows, and is not an admission that any of the work described is priorart to the claimed invention.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions designed todecrease intraocular pressure (IOP) of the eye. The compositions of theinvention can be used in the preparation of a medicament for thetreatment of eye conditions displaying increased IOP such as, forexample, glaucoma, infection, inflammation, uveitis, and diabeticretinopathy.

The compositions of the invention comprise short interfering nucleicacid molecules (siNA) that decrease or inhibit expression of genesassociated with production or drainage of intraocular fluid. In oneembodiment, siNAs of the invention decrease or inhibit expression ofgenes that are associated with production of intraocular fluid (e.g.,aqueous humor). Examples of such genes that are targets of the inventioninclude, but not limited to, Carbonic Anhydrases II, IV and XII;Adrenergic Receptors: beta 1 and 2 and alpha 1A, 1B and 1D; and ATPases:alpha 1, alpha 2, alpha 3, beta 1, beta 2. In another embodiment of theinvention, siNAs of the invention decrease or inhibit expression ofgenes associated with drainage of intraocular fluid (e.g., aqueoushumor). Examples of such genes that are targets of the inventioninclude, but not limited to Acetylcholinesterase; ProstaglandinEndoperoxide Synthases 1 and 2; Selectin E; Angiotensin System:Angiotensin II, Angiotensin II Converting Enzymes (ACE I and ACE II),Angiotensin II Receptors (ATR1 and ATR2) and Renin; and Cochlin.

The present invention encompasses compositions and methods of use ofshort interfering nucleic acid (siNA) including, but not limited to,short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), and short hairpin RNA (shRNA) molecules capable of mediatingRNA interference (RNAi) against the target genes identified supra. Inpreferred embodiments, the siNA used in the methods of the invention aredsRNA. siNAs of the invention can be unmodified or chemically-modified.

The methods of the invention comprise the administration to a patient inneed thereof of an effective amount of one or more siNAs of theinvention. In embodiments where more than one type of siNA isadministered, the siNAs can all be directed against the same ordifferent target genes. In preferred embodiments, the methods of theinvention provide a sustained decrease in IOP when compared with theduration of IOP decrease that results from administration ofcommercially available drugs (e.g., Xalatan, Trusopt, and Timoftol).

Methods of the invention also encompass administration of one or moresiNAs of the invention in combination with one or more othertherapeutics that decrease IOP including, but not limited to,commercially available drugs (e.g., Xalatan, Trusopt, and Timoftol).

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the GenBank Accession Numbers corresponding to the selectedhuman target genes.

FIGS. 2A-2MMMMM shows oligonucleotide sequences for siRNA moleculesencompassed by the present invention. “CA4” indicates carbonic anhydraseIV, “CA2” indicates carbonic anhydrase II, “CA12” indicates carbonicanhydrase XII, “ADRB I” indicates adrenergic, beta-1-, receptor; “ADRB2”indicates adrenergic, beta-2-, receptor; “ACHE” indicatesacetylcholinesterase; “SELF” indicates selectin E; “PTGS1” indicatesprostaglandin endoperoxide synthase 1; “PTGS2” indicates prostaglandinendoperoxide synthase 2; “ADRA1A” indicates adrenergic, alpha-IA-,receptor; “ADRA1B” indicates adrenergic, alpha-1B-, receptor; “ADRA1D”indicates adrenergic, alpha-1D-, receptor; “AGT” indicatesangiotensinogen; “AGTR1” indicates angiotensin II receptor, type 1;“AGTR2” indicates angiotensin II receptor, type 2; “ACE1” indicatesangiotensin I converting enzyme 1; “ACE2” indicates angiotensin Iconverting enzyme 2; “REN” indicates renin; “COCH” indicates coagulationfactor C homolog (cochlin); “ATP1A1” indicates ATPase, Na+/K+transporting, alpha 1 polypeptide; “ATP1A2” indicates ATPase, Na+/K+transporting, alpha 2 (+) polypeptide; “ATP1A3” indicates ATPase, Na+/K+transporting, alpha 3 polypeptide; “ATP1B1” indicates ATPase, Na+/K+transporting, beta 1 polypeptide; ATP1B2” indicates ATPase, Na+/K+transporting, beta 2 polypeptide.

FIGS. 3A-3F shows selected oligonucleotide sequences for siRNA moleculestested in in vitro in human OMDC cells (“VTH”), in vitro in rabbit NPEcells (“VTR”), and/or in vivo (“VV”) experiments. All sequences arehuman unless otherwise specified (“Hom, to” indicates a rabbit sequencethat is homologous to the indicated human sequence). SEQ IDNOS:1830-1833 are rabbit sequences with no corresponding disclosed humansequence.

FIGS. 4A-4B shows the effect of siRNA on gene expression in an in vitrosystem. RNA was prepared from cells treated with an siRNA molecule andanalyzed by semi-quantitative PCR. Semi-quantitative gels demonstratingexpression for (A) the adrenergic, beta-2-, receptor (siRNA were SEQ IDNOs: 122 in lane 1, 125 in lane 2, and 139 in lane 3) or (B)acetylcholinesterase (siRNA were SEQ ID NOS: 162 in lane 1 and 167 inlane 2) are shown. Lower panels show levels of beta actin in the cellsas a control. Lanes are as follows: M=molecular weight marker, C=controlcells, TC=transfection control cells, 1-3=the different siRNAs used toinhibit expression, NC=negative control. “30c” indicates 30 PCR cyclesand “40c” indicates 40 PCR cycles.

FIG. 5 shows the effect of inhibiting carbonic anhydrase II or carbonicanhydrase IV on IOP levels in rabbits in vivo. siRNA molecules targetingrabbit sequences for carbonic anhydrase II (SEQ ID NO:1838; homologousto SEQ ID NO:73) and carbonic anhydrase IV (SEQ ID NO:5) were tested inan in vivo rabbit model. A 256 μg dose of the indicated siRNA wasadministered at time points indicated by an arrow. SEQ ID NO:73decreased IOP by 25% while SEQ ID NO:5 decreased IOP by 16% over asaline control.

FIG. 6 shows the effect of inhibiting the adrenergic, beta-1-, receptoror the adrenergic, beta-2-, receptor on IOP levels in rabbits in vivo.siRNA molecules targeting rabbit sequences for the adrenergic, beta-1-,receptor (SEQ ID NO: 105) and the adrenergic, beta-2-, receptor (SEQ IDNO:1841; homologous to SEQ ID NO:139) were tested in an in vivo rabbitmodel. A 256 μg dose of the indicated siRNA was administered at timepoints indicated by an arrow. SEQ ID NO:105 decreased IOP by 25% whileSEQ ID NO:139 decreased IOP by 22% over a saline control.

FIG. 7 shows the effect of inhibiting acetylcholinesterase on IOP levelsin rabbits in vivo. A siRNA molecule targeting rabbit sequence foracetylcholinesterase (SEQ ID NO:1846; homologous to SEQ ID NO: 189) wastested in an in vivo rabbit model. A 256 μg dose of the siRNA wasadministered at time points indicated by an arrow. SEQ ID NO:189decreased IOP by 25% over a saline control.

FIG. 8 shows the effect of inhibiting prostaglandin endoperoxidesynthase 2 on IOP levels in rabbits in vivo. A siRNA molecule targetingrabbit sequence for a prostaglandin endoperoxide synthase 2 (SEQ ID NO:426) was tested in an in vivo rabbit model. A 256 μg dose of the siRNAwas administered at time points indicated by an arrow. SEQ ID NO:426decreased IOP by 22% over a saline control.

FIGS. 9A-9D show the effect of inhibiting various molecules to decreaseproduction or increase the drainage of intraocular fluid on IOP levelsin rabbits in vivo. A siRNA molecule targeting either the human orrabbit sequence for the indicated target was tested in an in vivo rabbitmodel. A 256 μg dose of the siRNA was administered at time pointsindicated by an arrow. The targets were (A) ATPase, Na+/K+ transporting,alpha 1 polypeptide (SEQ ID NO: 1399), (B) ATPase, Na+/K+ transporting,beta 2 polypeptide (SEQ ID NO: 1820), (C) rabbit sequence of selectin E(SEQ ID NO:1848; homologous to SEQ ID NO: 262), (D) carbonic anhydraseXII (SEQ ID NO: 522). Effect of siRNAs are compared to saline controls.

FIG. 10 shows the dose dependent effect of inhibiting carbonic anhydraseII on IOP levels in rabbits in vivo. A siRNA molecule targeting therabbit sequence for a carbonic anhydrase II (SEQ ID NO:1838; homologousto SEQ ID NO:73) was tested in an in vivo rabbit model. Either a 256 μgdose, a 132.5 μg dose, or a 66.25 μg dose of the siRNA was administeredat time points indicated by an arrow.

FIG. 11 shows the effect of inhibiting the adrenergic, beta-2-, receptorwith consecutive applications of siRNA on IOP levels in rabbits in vivo.A siRNA molecule targeting the rabbit sequence for the adrenergic,beta-2-, receptor (SEQ ID NO:1841; homologous to SEQ ID NO: 139) wastested in an in vivo rabbit model. A 256 μg dose of the siRNA wasadministered at time points indicated by an arrow. Effect of siRNA iscompared to a saline control.

FIG. 12 shows the maximum decrease in IOP obtained in the rabbit in vivomodel using the indicated siRNAs or commercially available drugs. siRNAmolecules targeting the rabbit sequences for carbonic anhydrase II (SEQID NO:1838; homologous to SEQ ID NO: 73), carbonic anhydrase IV (SEQ IDNO: 5), the adrenergic, beta-2-, receptor (SEQ ID NO:1841; homologous toSEQ ID NO: 139), the adrenergic, beta-1-, receptor (SEQ ID NO: 105),acetylcholinesterase (SEQ ID NO:1846; homologous to SEQ ID NO:189),prostaglandin endoperoxide synthase 2 (SEQ ID NO:426) were administeredin four doses of 256 μg each. The commercially available drugs Trusopt,Timoftol and Xalatan were administered in four doses of 8 mg, 1 mg, or20 μg, respectively.

FIG. 13 shows a comparison of the effect of decreasing aqueous humorproduction with increasing drainage rate on IOP levels in rabbits invivo. Aqueous humor production was decreased by inhibiting carbonicanhydrase II with siRNA and drainage rate was increased with theprostaglandin analog Xalatan. A 265 μg dose of either a siRNA moleculetargeting the rabbit sequence for carbonic anhydrase II (SEQ ID NO:1838;homologous to SEQ ID NO: 73) or a 20 μg dose of the drug Xalatan wereadministered at time points indicated by an arrow to an in vivo rabbitmodel.

FIG. 14 shows a comparison in length of action of various siRNAtreatments with commercially available drugs on IOP levels in rabbits invivo. siRNA molecules targeting the rabbit sequences for carbonicanhydrase II (SEQ ID NO:1838; homologous to SEQ ID NO: 73), carbonicanhydrase IV (SEQ ID NO: 5), and the adrenergic, beta-2-, receptor (SEQID NO:1841; homologous to SEQ ID NO: 139) were administered in fourdoses of 256 μg each. The commercially available drugs Trusopt, Xalatan,and Timoftol were administered in four doses of 8 mg, 20 μg, or 1 mg,respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions that decreaseintraocular pressure (IOP) of the eye. The compositions of the inventioncomprise short interfering nucleic acid molecules (siNA) that decreaseexpression of genes associated with production or drainage ofintraocular fluid (e.g., aqueous humor). The compositions of theinvention can be used in the preparation of a medicament for thetreatment of an eye conditions displaying increased IOP such asglaucoma, infection, inflammation, uveitis, and diabetic retinopathy.The methods of the invention comprise the administration to a patient inneed thereof an effective amount of one or more siNAs of the invention.

5.1 Design of siNAs

siNAs of the invention are designed to modulate the activity bydecreasing or inhibiting the expression of target genes that affect IOP.In one embodiment, a decrease in or inhibition of the target geneexpression decreases the production of intraocular fluid (e.g., aqueoushumor). Examples of such target genes are Carbonic Anhydrase II,Carbonic Anhydrase IV, Carbonic Anhydrase XII, Adrenergic Receptor beta1, Adrenergic Receptor beta 2, Adrenergic Receptor alpha 1A, AdrenergicReceptor alpha 1B, Adrenergic Receptor alpha 1D, ATPase alpha 1, ATPasealpha 2, ATPase alpha 3, ATPase beta 1, and ATPase beta 2. In anotherembodiment, a decrease in or inhibition of the target gene expressionincreases the drainage of intraocular fluid (e.g., aqueous humor).Examples of such target genes are Acetylcholinesterase, Selectin E,Angiotensin II, Angiotensin II Converting Enzyme I, Angiotensin IIConverting Enzyme II, Angiotensin II Receptor 1, Angiotensin II Receptor2, Renin, Cochlin, Prostaglandin Endoperoxide Synthase 1, andProstaglandin Endoperoxide Synthase 2. GenBank Accession numbers forpreferred target genes are shown in FIG. 1.

A gene is “targeted” by a siNA according to the invention when, forexample, the siNA molecule selectively decreases or inhibits theexpression of the gene. The phrase “selectively decrease or inhibit” asused herein encompasses siNAs that affects expression of one gene aswell those that effect the expression of more than one gene. In caseswhere an siNA affects expression of more than one gene, the gene that istargeted is effected at least two times, three times, four times, fivetimes, ten times, twenty five times, fifty times, or one hundred timesas much as any other gene. Alternatively, a siNA targets a gene when thesiNA hybridizes under stringent conditions to the gene transcript. siNAscan be tested either in vitro or in vivo for the ability to target agene.

A short fragment of the target gene sequence (e.g., 19-40 nucleotides inlength) is chosen as the sequence of the siNA of the invention. In oneembodiment, the siNA is a siRNA. In such embodiments, the short fragmentof target gene sequence is a fragment of the target gene mRNA. Inpreferred embodiments, the criteria for choosing a sequence fragmentfrom the target gene MRNA to be a candidate siRNA molecule include 1) asequence from the target gene MRNA that is at least 50-100 nucleotidesfrom the 5′ or 3′ end of the native mRNA molecule, 2) a sequence fromthe target gene mRNA that has a G/C content of between 30% and 70%, mostpreferably around 50%, 3) a sequence from the target gene mRNA that doesnot contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC,GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessiblein the MRNA, and 5) a sequence from the target gene mRNA that is uniqueto the target gene. The sequence fragment from the target gene mRNA maymeet one or more of the criteria identified supra. In embodiments wherea fragment of the target gene mRNA meets less than all of the criteriaidentified supra, the native sequence may be altered such that the siRNAconforms with more of the criteria than does the fragment of the targetgene mRNA. In preferred embodiments, the siRNA has a G/C/content below60% and/or lacks repetitive sequences.

In some embodiments, each of the siNAs of the invention targets onegene. In one specific embodiment, the portion of the siNA that iscomplementary to the target region is perfectly complementary to thetarget region. In another specific embodiment, the portion of the siNAthat is complementary to the target region is not perfectlycomplementary to the target region. siNA with insertions, deletions, andpoint mutations relative to the target sequence are also encompassed bythe invention. Thus, sequence identity may calculated by sequencecomparison and alignment algorithms known in the art (see Gribskov andDevereux, Sequence Analysis Primer, Stockton Press, 1991, and referencescited therein) and calculating the percent difference between thenucleotide sequences by, for example, the Smith-Waterman algorithm asimplemented in the BESTFIT software program using default parameters(e.g., University of Wisconsin Genetic Computing Group). Greater than90%, 95%, or 99% sequence identity between the siNA and the portion ofthe target gene is preferred. Alternatively, the complementarity betweenthe siNA and native RNA molecule may be defined functionally byhybridization. A siNA sequence of the invention is capable ofhybridizing with a portion of the target gene transcript under stringentconditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or70° C. hybridization for 12-16 hours; followed by washing). A siNAsequence of the invention can also be defined functionally by itsability to decrease or inhibit the expression of a target gene. Theability of a siNA to effect gene expression can be determinedempirically either in vivo or in vitro.

In addition to siNAs which specifically target only one gene, degeneratesiNA sequences may be used to target homologous regions of multiplegenes. WO2005/045037 describes the design of siNA molecules to targetsuch homologous sequences, for example by incorporating non-canonicalbase pairs, for example mismatches and/or wobble base pairs, that canprovide additional target sequences. In instances where mismatches areidentified, non-canonical base pairs (for example, mismatches and/orwobble bases) can be used to generate siNA molecules that target morethan one gene sequence. In a non-limiting example, non-canonical basepairs such as UU and CC base pairs are used to generate siNA moleculesthat are capable of targeting sequences for differing targets that sharesequence homology. As such, one advantage of using siNAs of theinvention is that a single siNA can be designed to include nucleic acidsequence that is complementary to the nucleotide sequence that isconserved between homologous genes. In this approach, a single siNA canbe used to inhibit expression of more than one gene instead of usingmore than one siNA molecule to target different genes.

Preferred siNA molecules of the invention are double stranded. In oneembodiment, double stranded siNA molecules comprise blunt ends. Inanother embodiment, double stranded siNA molecules comprise overhangingnucleotides (e.g., 1-5 nucleotide overhangs, preferably 2 nucleotideoverhangs). In a specific embodiment, the overhanging nucleotides are 3′overhangs. In another specific embodiment, the overhanging nucleotidesare 5′ overhangs. Any type of nucleotide can be a part of the overhang.In one embodiment, the overhanging nucleotide or nucleotides areribonucleic acids. In another embodiment, the overhanging nucleotide ornucleotides are deoxyribonucleic acids. In a preferred embodiment, theoverhanging nucleotide or nucleotides are thymidine nucleotides. Inanother embodiment, the overhanging nucleotide or nucleotides aremodified or non-classical nucleotides. The overhanging nucleotide ornucleotides may have non-classical internucleotide bonds (e.g., otherthan phosphodiester bond).

In preferred embodiments, siNA compositions of the invention are any ofSEQ ID NOS:1-1829. In an even more preferred embodiments, dsRNAcompositions of the invention are any of SEQ ID NOS:1-1829 hybridized toits complement. The invention also encompasses siNAs that are 40nucleotides or less and comprise a nucleotide sequence of any of SEQ IDNOS:1-1829 as well as dsRNA compositions that are 40 nucleotides or lessand comprise a nucleotide sequence of any of SEQ ID NOS:1-1829hybridized to its complement. In a specific embodiment, the siNA is21-30 nucleotides and comprises any one of SEQ ID NOS:1-1829.

5.2 Synthesis of siNAs

siNAs designed by methods described in Section 5.1 can be synthesized byany method known in the art. RNAs are preferably chemically synthesizedusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Additionally, siRNAs can be obtainedfrom commercial RNA oligo synthesis suppliers, including, but notlimited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,Colo., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland,Mass., USA), and Cruachem (Glasgow, UK), Qiagen (Germany), Ambion (USA)and Invitrogen (Scotland). Alternatively, siNA molecules of theinvention can be expressed in cells by transfecting the cells withvectors containing the reverse complement siNA sequence under thecontrol of a promoter. Once expressed, the siNA can be isolated from thecell using techniques well known in the art.

In embodiments where the siRNA is a dsRNA, an annealing step isnecessary if single-stranded RNA molecules are obtained. Briefly,combine 30 μl of each RNA oligo 50 μM solution in 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate. The solution isthen incubated for 1 minute at 90° C., centrifuged for 15 seconds, andincubated for 1 hour at 37° C.

In embodiments where the siRNA is a short hairpin RNA (shRNA); the twostrands of the siRNA molecule may be connected by a linker region (e.g.,a nucleotide linker or a non-nucleotide linker).

5.3 Chemical Modification of siNAs

The siNAs of the invention may contain one or more modified nucleotidesand/or non-phosphodiester linkages. Chemical modifications well known inthe art are capable of increasing stability, availability, and/or celluptake of the siNA. The skilled person will be aware of other types ofchemical modification which may be incorporated into RNA molecules (seeInternational Publications WO03/070744 and WO2005/045037 for an overviewof types of modifications).

In one embodiment, modifications can be used to provide improvedresistance to degradation or improved uptake. Examples of suchmodifications include phosphorothioate internucleotide linkages,2′-O-methyl ribonucleotides (especially on the sense strand of a doublestranded siRNA), 2′-deoxy-fluoro ribonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides,and inverted deoxyabasic residue incorporation (see generallyGB2406568).

In another embodiment, modifications can be used to enhance thestability of the siRNA or to increase targeting efficiency.Modifications include chemical cross linking between the twocomplementary strands of an siRNA, chemical modification of a 3′ or 5′terminus of a strand of an siRNA, sugar modifications, nucleobasemodifications and/or backbone modifications, 2′-fluoro modifiedribonucleotides and 2′-deoxy ribonucleotides (see generallyInternational Publication WO2004/029212).

In another embodiment, modifications can be used to increased ordecreased affinity for the complementary nucleotides in the target mRNAand/or in the complementary siNA strand (see generally InternationalPublication WO2005/044976). For example, an unmodified pyrimidinenucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or5-propynyl pyrimidine. Additionally, an unmodified purine can besubstituted with a 7-deza, 7-alkyl, or 7-alkenyl purine.

In another embodiment, when the siNA is a double-stranded siRNA, the3′-terminal nucleotide overhanging nucleotides are replaced bydeoxyribonucleotides (see generally Elbashir et al., 2001, Genes Dev,15:188).

5.4 Demonstration of Therapeutic Utility

The compositions and methods of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic activity prior touse in humans. For example, in vitro assays which can be used todetermine whether administration of a specific therapeutic protocol isindicated, include in vitro cell culture assays in which a candidatesiNA is administered to cells (e.g., rabbit non-pigmented cilliaryepithelium cells (NPE), human cilliary epithelium cells (OMDC), or humanembryonic kidney cells (HEK293)) in vitro and the effect of suchprotocol upon the cells is observed, e.g., decreased or inhibitedexpression of the target gene.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrabbits, rats, mice, chicken, cows, monkeys, hamsters, etc. For example,the New Zealand rabbit is the preferred standard in experimentalplatforms designed to study IOP. It is easy to handle and it has a bigeye, similar in size to the human organ. In addition, present equipmentto measure IOP is not suited to use in animals with small eyes such asmice or rats. Finally, rabbits have an IOP (about or equal to 23 mm Hg)that can be reduced to 40% of its normal (or pre-drug) value (e.g., toabout or equal to 9 mm Hg) using local commercial hypotensivemedication. Thus, although it is possible to generate rabbit glaucomamodels (for example, surgically blocking episclerotic veins orartificially occluding the trabecular meshwork), generally those in thefield use normotensive rabbits.

5.5 Therapeutic Methods

The present invention encompasses methods for treating, preventing, ormanaging an eye disorder associated with increased IOP in a patient(e.g., a mammal, especially humans) comprising administering aneffective amount of one or more siNAs of the invention. In a specificembodiment, the disorder to be treated, prevented, or managed isglaucoma. Any type of glaucoma that is associated with IOP can betreated with the methods of the present invention including, but notlimited to, Open Angle Glaucoma (e.g., Primary Open Angle Glaucoma,Pigmentary Glaucoma, and Exfoliative Glaucoma, Low Tension Glaucoma),Angle Closure Glaucoma (also known clinically as closed angle glaucoma,narrow angle glaucoma, pupillary block glaucoma, and ciliary blockglaucoma) (e.g., Acute Angle Closure Glaucoma and Chronic Angle ClosureGlaucoma), Aniridic Glaucoma, Congenital Glaucoma, Juvenile Glaucoma,Lens-Induced Glaucoma, Neovascular Glaucoma, Post-Traumatic Glaucoma,Steroid-Induced Glaucoma, Sturge-Weber Syndrome Glaucoma, andUveitis-Induced Glaucoma.

In preferred embodiments, the siNAs used in the therapeutic methods ofthe invention decrease or inhibit the expression of genes that effectIOP, for example, Carbonic Anhydrase II, Carbonic Anhydrase IV, CarbonicAnhydrase XII, Adrenergic Receptor beta 1, Adrenergic Receptor beta 2,Adrenergic Receptor alpha 1A, Adrenergic Receptor alpha 1B, AdrenergicReceptor alpha 1D, ATPase alpha 1, ATPase alpha 2, ATPase alpha 3,ATPase beta 1, and ATPase beta 2, Acetylcholinesterase, Selectin E,Angiotensin II, Angiotensin II Converting Enzyme I, Angiotensin IIConverting Enzyme II, Angiotensin II Receptor 1, Angiotensin II Receptor2, Renin, Cochlin, Prostaglandin Endoperoxide Synthase 1, andProstaglandin Endoperoxide Synthase 2. In certain embodiments, one ormore of the siNAs of the invention are selected from the groupconsisting of SEQ ID NOS:1-1829. In a specific preferred embodiment, thesiNAs used in the therapeutic methods of the invention are dsRNA of anyof SEQ ID NOS:1-1829 hybridized to its complement. The invention alsoencompasses siNAs that are 40 nucleotides or less and comprise anucleotide sequence of any of SEQ ID NOS:1-1829 as well as dsRNAcompositions that are 40 nucleotides or less and comprise a nucleotidesequence of any of SEQ ID NOS:1-1829 hybridized to its complement. In aspecific embodiment, the siNA is 21-30 nucleotides and comprises any onof SEQ ID NOS:1-1829.

In preferred embodiments, the methods of the invention provide asustained decrease in IOP that lasts for longer than 8, 10, 12, or 14hours, more preferably for several days (e.g., 2 days, 3 days, 4 days,or 5 days), after the last administration of siNA. In such embodiments,the effect (i.e., decreased IOP) of administered siNAs of the inventionis longer lasting than the duration of IOP decrease that typicallyresults from administration of presently commercially available drugs(e.g., Xalatan, Trusopt, and Timoftol). The siNAs of the invention thatprovide sustained IOP decreasing action can be administered in a regimensuch that IOP is continually decreased without daily administration ofthe siNA. In a specific embodiment, a treatment regimen can includeconsecutive cycles of administration (e.g., one dose of siNA given dailyfor four days) and non-administration (e.g., 3 or 4 days with notreatment given) while still eliciting a continual decrease in IOP.

In one embodiment, a single type of siNA is administered in thetherapeutic methods of the invention. In another embodiment, an siNA ofthe invention is administered in combination with another siNA of theinvention and/or with one or more other non-siNA therapeutic agentsuseful in the treatment, prevention or management of an eye disorderassociated with increased IOP. The term “in combination with” is notlimited to the administration of therapeutic agents at exactly the sametime, but rather it is meant that the siNAs of the invention and theother agent are administered to a patient in a sequence and within atime interval such that the benefit of the combination is greater thanthe benefit if they were administered otherwise. For example, eachtherapeutic agent may be administered at the same time or sequentiallyin any order at different points in time; however, if not administeredat the same time, they should be administered sufficiently close in timeso as to provide the desired therapeutic effect. Each therapeutic agentcan be administered separately, in any appropriate form and by anysuitable route.

5.6 Dosage

As used herein, an “effective amount” refers to that amount of a siNA ofthe invention sufficient to treat or manage an eye disorder associatedwith increased IOP and, preferably, the amount sufficient to decreaseIOP. For treatment of increased IOP in humans, it is preferred to reduceIOP so that IOP is between about 14 and 20 mm Hg. However, any reductionin IOP as compared to pretreatment IOP is advantageous (e.g., a decreasein IPO greater that 5%, 10%, 25%, 30%, 35%, 40%, 50%, or 60% ofpretreatment IOP). A therapeutically effective amount may also refer tothe amount of an siNA sufficient to delay or minimize the onset of aneye disorder associated with IOP. A therapeutically effective amount mayalso refer to the amount of the therapeutic agent that provides atherapeutic benefit in the treatment or management of an eye disorderassociated with IOP. Further, a therapeutically effective amount withrespect to an siNA of the invention means that amount of therapeuticagent alone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of an eye disorderassociated with IOP. Used in connection with an amount of an siRNA ofthe invention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapeutic agent. Treatment withsiNA alone or in combination should result in an IOP of about 14 and 20mm Hg. However, any decrease in IOP as compared to pretreatment IOP isadvantageous (e.g., a decrease in IPO greater that 5%, 10%, 25%, 30%,35%, 40%, 50%, or 60% of pretreatment IOP).

The effective amount of a composition of the invention can be determinedby standard research techniques. For example, the dosage of thecomposition which will be effective in the treatment, prevention ormanagement of the disorder can be determined by administering thecomposition to an animal model such as, e.g., the animal modelsdisclosed herein or known to those skilled in the art. In addition, invitro assays may optionally be employed to help identify optimal dosageranges. Alternatively, the dosage may be determined for an individual bytitrating the dose until an effective level is reached.

Selection of the preferred effective amount to be used in dosages can bedetermined (e.g., via clinical trials) by a skilled artisan based uponthe consideration of several factors which will be known to one ofordinary skill in the art. Such factors include the disorder to betreated or prevented, the symptoms involved, the patient's body mass,the patient's immune status and other factors known by the skilledartisan to reflect the accuracy of administered pharmaceuticalcompositions.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

When the siRNA is administered directly to the eye, generally an amountof between 0.3 mg/kg-20 mg/kg, 0.5 mg/kg-10 mg/kg, or 0.8 mg/kg-2 mg/kgbody weight/day of siNA is administered. When the siRNA is administeredintravenously, generally an amount of between 0.5 mg-20 mg, or 0.8 mg-10mg, or 1.0 mg-2.0 mg/injection is administered.

5.7 Formulations and Routes of Administration

The siNAs of the invention may be formulated into pharmaceuticalcompositions by any of the conventional techniques known in the art (seefor example, Alfonso, G. et al., 1995, in: The Science and Practice ofPharmacy, Mack Publishing, Easton Pa., 19th ed.). Formulationscomprising one or more siNAs for use in the methods of the invention maybe in numerous forms, and may depend on the various factors specific foreach patient (e.g., the type and severity of disorder, type of siNAadministered, age, body weight, response, and the past medical historyof the patient), the number and type of siNAs in the formulation, theform of the composition (e.g., in liquid, semi-liquid or solid form),the therapeutic regime (e.g. whether the therapeutic agent isadministered over time as a slow infusion, a single bolus, once daily,several times a day or once every few days), and/or the route ofadministration (e.g., topical, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, or sublingual means).

These compositions can take the form of aqueous and non aqueoussolutions, suspensions, emulsions, microemulsions, aqueous and nonaqueous gels, creams, tablets, pills, capsules, powders,sustained-release formulations and the like. The siNAs of the inventioncan also be encapsulated in a delivery agent (including, but not limitedto, liposomes, microspheres, microparticles, nanospheres, nanoparticles,biodegradable polymers, hydrogels, cyclodextrins poly(lactic-co-glycolic) acid (PLGA)) or complexed with polyethyleneimineand derivatives thereof (such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives).

Pharmaceutical carriers, vehicles, excipients, or diluents may beincluded in the compositions of the invention including, but not limitedto, water, saline solutions, buffered saline solutions, oils (e.g.,petroleum, animal, vegetable or synthetic oils), starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, ethanol, biopolymers (e.g., carbopol,hialuronic acid, polyacrylic acid, etc.), dextrose, permeation enhancers(e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids),and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone)and the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. In addition,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyloleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

Pharmaceutical compositions can be administered systemically or locally,e.g., near the intended site of action (i.e., the eye). Additionally,systemic administration is meant to encompass administration that cantarget to a particular area or tissue type of interest.

In preferred embodiments, the compositions of the invention areformulated in a solution or a gel for topical administration to the eye.In such embodiments, the formulations may be cationic emulsions and/orcontain biopolymers including, but not limited to,poly(lactide-co-glycolide), carbopol, hialuronic acid and polyacrylicacid.

The siNAs of the present invention can also be formulated in combinationwith other therapeutic compounds that decrease IOP (e.g., commerciallyavailable drugs).

Alternatively, the siNAs can be expressed directly in cells of interest(e.g., the eye, more particularly cells of the trabecular meshwork orpigmented cilliary epithelium cells) by transfecting the cells withvectors containing the reverse complement siNA sequence under thecontrol of a promoter. For double stranded siNAs, cells can betransfected with one or more vectors expressing the reverse complementsiNA sequence for each strand under the control of a promoter. The cellof interest will express the siNA directly without having to beadministered a composition of the invention.

The contents of all published articles, books, reference manuals andabstracts cited herein, are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Modifications and variations ofthe present invention are possible in light of the above teachings.

6. EXAMPLES 6.1 Design of siRNAs

For each gene target, several siNA molecules were designed usingproprietary software. The proprietary software used a number of criteriain choosing a sequence fragment of a gene as a candidate siRNA moleculeincluding 1) a sequence from the target gene MRNA that is at least50-100 nucleotides from the 5′ or 3′ end of the native MRNA molecule, 2)a sequence from the target gene mRNA that has a G/C content of between30% and 70%, most preferably around 50%, 3) a sequence from the targetgene mRNA that does not contain repetitive sequences (e.g., AAA, CCC,GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target genemRNA that is accessible in the mRNA, and 5) a sequence from the targetgene mRNA that is unique to the target gene.

Briefly, each of the target genes was introduced as a nucleotidesequence in a prediction program that takes into account all thevariables described supra for the design of optimal oligonucleotides foruse as siRNA. This program scanned any mRNA nucleotide sequence forregions susceptible to be targeted by siRNAs and thus were goodcandidates for use as the sequence of the siRNA molecule itself. Theoutput of this analysis was a score of possible siRNA oligonucleotides.The highest scores were used to design double stranded RNAoligonucleotides (typically 19 bp long) that were typically made bychemical synthesis.

Target genes are listed with their GenBank Accession numbers in FIG. 1.siRNA molecules directed to the target genes are listed in FIG. 2 FIGS.2A-2MMMMM. All siRNA molecules used in the experiments described infrawere designed to have a 2 thymidine nucleotide 3′ overhang. Some of thesiRNA molecules were designed to target rabbit homologs of human targetgenes in preparation for in vivo assays in a rabbit model. Those siRNAstargeting rabbit genes are identified in FIGS. 3A-3F with “Hom, to”indicating the human siRNA that each is homologous to. Specifically, SEQID NOS:1834-1862 are rabbit homolog of human sequences. Four siRNAstested, SEQ ID NOS:1830-1833, do not have a corresponding humanhomologous sequence.

6.2 In Vitro Assays

Cells (either NPE, OMDC, or HEK293 cells) were incubated with varioussiRNA molecules and assayed for expression of the native mRNAcorresponding to the siRNA molecule. One day prior to transfection,2-4×10⁵ cells were seeding into each well of a 6 well plate in 3 ml ofgrowth medium (DMEM, 10% serum, antibiotics and glutamine) and incubatedunder normal growth conditions (37° C. and 5% CO₂). Lipofectamine 2000Reagent (Invitrogen Corporation, Carlsbad, Calif.) was used to transfectthe cells with the siRNA molecules. The protocol supplied by themanufacturer was followed. Briefly, siRNA molecules were added to cellsthat were 30%-50% confluent to a final concentration of 100 nM. Prior toaddition to the cells, the siRNA molecule was diluted in 250 μl DMEM andincubated at room temperature for 5 minutes. The siRNA was then mixedwith 6 μl of Lipofectamine 2000 Reagent that also had been diluted in250 μl DMEM and the mixture was incubated at room temperature for 20minutes. The siRNA/Lipofectamine mixture was added to the cellsdrop-wise with 2 ml of fresh growth medium low in antibiotics. Afterswirling the plates to ensure uniform distribution of the transfectioncomplexes, the cells were incubated under normal growth conditions for24 hours. After incubation with either of the transfection complexes,the medium was removed and replaced with 3 ml of fresh complete growthmedium. mRNA was collected from cells at 24, 48 and 72 hourspost-transfection.

After transfection and incubation with a siRNA molecule, total RNAfractions were extracted from cells using protocols well known in theart. The effect of siRNAs on target gene expression was analyzed by realtime PCR and semi-quantitative PCR according to standard protocols.Approximately 250 ng of total RNA was used for reverse transcriptionfollowed by PCR amplification with specific primers for the target genein a reaction mixture containing SYBR Green I Dye (Applied Biosystems,Foster City, Calif.). Basic PCR conditions comprised an initial step of30 minutes at 91° C., followed by 40 cycles of 5 s at 95° C., 10 s at62° C. and 15 s at 72° C. Quantification of beta-actin mRNA was used asa control for data normalization.

Table 1 shows representative results of real time PCR experiments forsome of the target genes. The values represent the mean of thepercentage of siRNA interference of each gene expression once normalizedwith the control cells and their standard deviations. Compared to thecontrol cells, the level of the different transcripts at both 24 and 48h time points was significantly reduced after the siRNA treatment.

TABLE 1 % of gene transcript level in control cells Target siRNA used 24h 48 h CA2 SEQ ID NO: 73 76.25 ± 12.60 84.57 ± 14.70 SEQ ID NO: 54 37.97± 9.78  61.45 ± 9.62  SEQ ID NO: 66 35.30 ± 9.73  51.14 ± 16.49 PTGS1SEQ ID NO: 353 42.25 ± 13.76 42.68 ± 17.00 SEQ ID NO: 369 34.98 ± 14.3326.30 ± 10.91 PTGS2 SEQ ID NO: 426 68.68 ± 12.48 70.17 ± 19.21 SEQ IDNO: 421 81.00 ± 13.54 66.85 ± 18.67 SEQ ID NO: 477 75.45 ± 14.71 61.83 ±16.96

FIG. 4 shows the effect of siRNA on gene expression for the adrenergic,beta-2-, receptor (FIG. 4A) and acetylcholinesterase (FIG. 4B). ThesiRNA molecules used for each were SEQ ID NOs: 122, 125, and 139 for theadrenergic, beta-2-, receptor (lanes 1-3 of FIG. 4A, respectively) andSEQ ID NOs: 162 and 167 for acetylcholinesterase (in lanes 1-2 of FIG.4B, respectively). Lower panels show levels of beta actin in the cellsas a control. “Control cells” were non-transfected NPE cells,“transfection control cells” were NPE cells transfected with an siRNAwith a scrambled sequence, and “negative control cells” were a PCRcontrol. Either 30 (indicated by “30c”) or 40 (indicated by “40c”) PCRcycles were run.

6.3 In Vivo Assays

Normotensive New Zealand White rabbits (males, 2-3 kg) were used in thein vivo assays. The animals were kept in individual cages with freeaccess to food and water. Animals were submitted to artificial 12 hourslight/darkness cycles to avoid uncontrolled circadian oscillations ofIOP and all experiments were performed at the same time of day controlfor any fluctuations in IOP due to circadian oscillations. Animalhandling and treatment were carried out in accordance with the EuropeanCommunities Council Directive (86/609/EEC) and the statement of theAssociation for Research in Vision and Ophthalmology on the Use ofAnimals in Ophthalmic and Vision Research. Each animal was used for onlyone experiment.

IOP measurements were done using a contact tonometer (TonoPen XL,Mentor, Norwell, Mass.) due to past success of measuring intraocularpressures within the range of 3 to 30 mm Hg in rabbits (Abrams et al.,1996, Invest Ophthalmol Vis Sci. 37:940-4). Measurements were performedby delicately applying the tonometer's sensor to the corneal surface ofthe animal. All measurements fell within the 3 to 30 mm Hg interval withthe mean baseline value of intraocular pressure being 17.0±0.39 mm Hgfor untreated animals (n=100). In order to avoid distress to the animal,rabbits were topically anesthetized (10 μl of oxibuprocaine/tetracaine,0.4%/1%, in a saline solution (1/4 v:v)) prior to IOP measurement.

Commercially available drugs were typically administered to the animalsby instilling a small volume (typically 40 μl) on the corneal surface.Contralateral eyes were treated with the vehicle alone and were used ascontrols in each experiment.

siRNA molecules or commercially available drugs were typicallyadministered to the animals as follows. Doses of siRNA in salinesolution (0.9% w/v) to a final volume of 40 μl were applied to thecorneal surface of one eye each day during four consecutive days. Theopposite eye was taken as a control and 40 μl of sterile saline (0.9%w/v) was instilled on it at the same time points. Commercially availabledrugs were typically administered to the animals by instilling a smallvolume (typically 40 μl) on the corneal surface. Contralateral eyes weretreated with the vehicle alone and were used as controls in eachexperiment. The IOP was measured before each application and at 2 h, 4 hand 6 h following the instillation for 10 days.

The data are summarized in Table 2 where values represent the mean ofthe normalized maximum percentage of IOP reduction after siRNA treatmentand their standard deviations. The decrease in IOP was statisticallysignificant for all the treated targets. These results indicated thatboth siRNAs and commercial drugs reduced IOP levels around 20%, althoughsiRNAs presented a more maintained effect. No secondary effects wereobserved in the animals during the experimental protocols.

TABLE 2 IOP reduction (% of Target siRNA Used saline control) CA2 SEQ IDNO: 1838 24.84 ± 3.41 (Hom. To SEQ ID NO: 73) CA4 SEQ ID NO: 5 14.47 ±5.00 CA12 SEQ ID NO: 522 24.30 ± 1.29 ADRB1 SEQ ID NO: 105 28.04 ± 2.98ADRB2 SEQ ID NO: 1841 21.18 ± 1.88 (Hom. To SEQ ID NO: 139) ADRAIA SEQID NO: 1856  9.51 ± 1.04 (Hom. To SEQ ID NO: 546) ADRAIB SEQ ID NO: 185817.48 ± 1.30 (Hom. To SEQ ID NO: 619) ACHE SEQ ID NO: 1846 25.25 ± 2.70(Hom. To SEQ ID NO: 189) PTGS1 SEQ ID NO: 1850 14.62 ± 1.93 (Hom. To SEQID NO: 322) PTGS2 SEQ ID NO: 426 23.78 ± 2.27 SELE SEQ ID NO: 1848 21.80± 1.74 (Hom. To SEQ ID NO: 262) ACE1 SEQ ID NO: 1860 17.51 ± 1.28 (Hom.To SEQ ID NO: 866) AGTR1 SEQ ID NO: 1859  9.72 ± 1.35 (Hom. To SEQ IDNO: 705) AGTR2 SEQ ID NO: 774 11.22 ± 1.53 ATP1A1 SEQ ID NO: 1399 18.13± 1.39 ATP1B2 SEQ ID NO: 1820 16.32 ± 0.91 Xalatan — 25.46 ± 5.24Trustop — 16.41 ± 2.38 * “Hom. To” indicates that the siRNA used was therabbit homolog of the indicated human sequence

FIGS. 5-9 show time course experiments over 10 days for the indicatedsiRNAs using the in vivo rabbit model of IOP. The indicated siRNA wasadministered one time on each of days 1-4 of the experiment. Maximumresponses (i.e., decrease in IOP) were generally observed on day 2 or 3of the experiment and lasted for several days.

FIG. 10 shows the dose dependent effect of inhibiting carbonic anhydraseII on IOP levels in the in vivo rabbit model. Either a 265 μg, 132.5 μg,or 66.25 μg dose of the indicated siRNA was administered on each of days1-4 of the ten day experiment. Although all levels of dose decreasedIOP, there was a greater degree of decrease with increasing amounts ofsiRNA used.

FIG. 11 shows the effect of inhibiting the adrenergic, beta-2-, receptorwith consecutive applications of siRNA on IOP levels in the in vivorabbit model. The indicated siRNA was administered one time on each ofdays 1-4, 7-10, and 15-18 of the twenty eight day experiment. Decreasedlevels of IOP were maintained with administration schemes at 3 dayintervals.

FIG. 12 shows a comparison of the maximum decrease in IOP in the in vivorabbit model using the indicated siRNAs and commercially availabledrugs. For the siRNAs and drugs that decrease aqueous humor production,all of the siRNAs elicited a maximum decrease in IOP greater than thatof Trusopt but less than that of Timoftol. For the siRNAs and drugs thatincreased drainage rate, all of the siRNAs elicited a maximum decreasein IOP greater than that of Xalatan.

FIG. 13 shows a time course experiment over 10 days for the indicatedsiRNA and drug using the in vivo rabbit model of IOP. The indicatedsiRNA or drug was administered one time on each of days 1-4 of theexperiment. Maximum responses (i.e., decrease in IOP) were generallyobserved on day 2 or 3 of the experiment for the siRNA but were moreimmediate for the drug. Although the drug acted more quickly than siRNAin decreasing IOP, it only maintained an effect for about 8 hourswhereas the effect of the siRNA lasted several days.

FIG. 14 shows a comparison in length of action of various siRNAtreatments with commercially available drugs on IOP levels in the invivo rabbit model. The indicated siRNA or drug was administered in fourdoses (one dose each on four consecutive days) and the IOP was measuredfour times a day during days 0-10. The Effect₅₀ represents the timeinterval between the moment when the IOP reaches a value which is 50% ofthe maximum decrease reached and the moment when the IOP level starts torecover to values higher than 50% of the maximum decrease value. All ofthe siRNAs tested decreased IOP for a longer period of time than any ofthe drugs.

1. A method of treating an eye disorder characterized by increasedintraocular pressure (IOP) comprising topically administering to thecorneal surface of the eye of a patient in need thereof a shortinterfering nucleic acid molecule (siNA) comprising a double-strandednucleic acid region, in an amount that reduces IOP in the eye of thepatient, wherein said siNA targets carbonic anhydrase IV, and whereinthere is greater than 90% sequence identity or greater than 90% sequencecomplementarity between said double-stranded nucleic acid region of siNAand the portion of mRNA encoding carbonic anhydrase IV that is targetedby said siNA.
 2. The method of claim 1 wherein the siNA provides asustained decrease in IOP that lasts for longer than 24 hours afteradministration of the siNA.
 3. The method of claim 1 wherein thedecrease in IOP is present for at least 8 hours.
 4. The method of claim1 wherein decreased IOP persists for at least 2 days.
 5. The method ofclaim 1 wherein the siNA is short interfering ribonucleic acid (siRNA).6. The method of claim 5 wherein the siRNA is double stranded (dsRNA).7. The method of claim 5 wherein the siRNA is short hairpin (shRNA). 8.The method of claim 1 wherein the siNA comprises at least one modifiedoligonucleotide.
 9. The method of claim 1 wherein the siNA comprises atleast one linkage between two nucleotides that is not a phosphodiesterlinkage.
 10. The method of claim 1 wherein the eye disorder is selectedfrom the group consisting of glaucoma, infection, inflammation, uveitis,and diabetic retinopathy.
 11. The method of claim 1, wherein saidportion of mRNA comprises the nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO:
 46. 12. The method ofclaim 1, wherein said portion of mRNA is 40 nucleotides or lesscomprising the nucleotide sequence selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO:
 46. 13. The method of any of claims 11 or 12wherein the siNA is hybridized to its complement to make a dsRNA. 14.The method of claim 13 wherein the dsRNA has a dinucleotide 3′ overhang.15. The method of claim 14 wherein the dinucleotide overhang is made ofthymidine nucleotides.
 16. The method of claim 1 wherein more than onetype of siNA is administered to the patient.
 17. The method of claim 16wherein the more than one type of siNA decreases or inhibits theexpression of the same gene.
 18. The method of claim 1, wherein there isgreater than 95% sequence identity or greater than 95% sequencecomplementarity between said double-stranded nucleic acid region of saidsiNA and the portion of mRNA encoding carbonic anhydrase IV that istargeted by said siNA.
 19. The method of claim 1, wherein there is 100%sequence identity or 100% sequence complementarity between saiddouble-stranded nucleic acid region of said siNA and the portion of mRNAencoding carbonic anhydrase IV that is targeted by said siNA.
 20. Themethod of claim 19, wherein the siNA is short interfering ribonucleicacid (siRNA) or double stranded ribonucleic acid (dsRNA).
 21. The methodof claim 19, wherein the eye disorder is selected from the groupconsisting of glaucoma, infection, inflammation, uveitis, and diabeticretinopathy.
 22. The method of claim 19, wherein the siNA comprises thenucleotide sequence selected from the group consisting of SEQ ID NO: 1to SEQ ID NO:
 46. 23. The method of claim 19, wherein the siNA is 40nucleotides or less comprising the nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO:
 46. 24. The method ofclaim 19, wherein the dsRNA has a dinucleotide 3′ overhang.
 25. Themethod of claim 24, wherein the dinucleotide overhang is made ofthymidine nucleotides.
 26. The method of claim 1, wherein said topicallyadministering consists of instilling said siNA molecule on said cornealsurface.
 27. The method of claim 1, wherein said siNA molecule isunmodified.